Isolation chip and manufacturing method of the same

ABSTRACT

A device, system, and method for separating, concentrating, and isolating target particles from a bioliquid sample includes a sample reservoir, a first filtration membrane, a second filtration membrane, a first chamber, and a second chamber. The first chamber with first outlet is connected to the sample reservoir through the first filtration membrane. The second chamber with second outlet is connected to the sample reservoir through the second filtration membrane. Negative pressures are applied alternately to the two filtration membranes to isolate target particles, clogging of the membranes being prevented by the same alternating but opposite-phase positive pressures.

FIELD

The subject matter herein generally relates to biotechnology, and moreparticularly, to an isolation chip and a manufacturing method of theisolation chip.

BACKGROUND

A biopsy of human liquid, such as urine, saliva, pleural effusion, andcerebrospinal liquid, is the sampling and analysis of the bioliquid.With isolation and study of specific biomarkers in the bioliquid, liquidbiopsy can be used as a diagnostic and monitoring tool for diseases suchas cancer, with the added benefit of being largely non-invasive. Thespecific biomarkers in the bioliquid include circulating tumor DNA(ctDNA), circulating tumor cells (CTCs), and microvesicles (i.e.exosomes). The study of CTCs and exosomes is helpful to obtaininformation from different perspectives, and thus improve the precisionof liquid biopsy.

The existing approaches to isolation and purification of CTCs andexosomes include centrifuging, testing immuno-affinities, and filtering.However, centrifuging may cause mechanical damages to CTCs and exosomes,and is limited in throughput for clinical applications. Immuno-affinityrelies on antibodies which results in higher cost, and the releaseprocess after immune-affinity may reduce the viability of CTCs andexosomes. Filtering is low cost and has high throughput, and thebiological sample after filtration has good viability. However, cloggingof the filtration membrane usually happens during filtration, which candecrease the isolation efficiency and purity of CTCs and exosomes.

Therefore, there is room for improvement in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Implementations of the present disclosure will now be described, by wayof embodiments only, with reference to the attached figures.

FIG. 1 is a diagrammatic view of an embodiment of an isolation chipaccording to the present disclosure.

FIG. 2 is an exploded view of the isolation chip of FIG. 1.

FIG. 3 is a flowchart of an embodiment of a manufacturing method of theisolation chip of FIG. 1.

FIG. 4 is a diagrammatic view of another embodiment of an isolationchip.

FIG. 5 is a flowchart of an embodiment of a manufacturing method of theisolation chip of FIG. 4.

FIG. 6 is a diagrammatic view of an embodiment of an isolation device.

FIG. 7 is a diagrammatic view showing liquid flow paths of the isolationdevice of FIG. 6.

FIG. 8 is a block diagram of an isolation control system of theisolation device of FIG. 6.

FIG. 9 is a diagrammatic view showing fluid paths in the isolation chipof FIG. 1 during sample isolation.

FIG. 10a is a diagram of an embodiment of negative pressure applied tothe isolation chip of FIG. 1.

FIG. 10b is a diagram of another embodiment of negative pressure appliedto the isolation chip of FIG. 1.

FIG. 10c is a diagram of yet another embodiment of negative pressureapplied to the isolation chip of FIG. 1.

FIG. 11 is a diagrammatic view of a mounting base of the isolationdevice of FIG. 3.

FIG. 12 is a diagrammatic view showing an isolation chip and air pipesmounted to the mounting base of FIG. 11.

FIG. 13 is a flowchart of an isolating method of target particles fromliquid sample.

FIG. 14a is a diagram showing a particle size distribution in anoriginal urine sample.

FIG. 14b is a diagram showing a particle size distribution of exosomesisolated from the urine sample by the isolation chip of FIG. 1.

FIG. 14c is a diagram showing a particle size distribution of exosomesisolated from the urine sample by qEV™ column (iZON Science).

FIG. 14d is a diagram showing a particle size distribution of exosomesisolated from the urine sample by ExoQuick-TC™ exosome precipitationreagent (SBI).

FIG. 14e is a diagram showing a particle size distribution of exosomesisolated from the urine sample by Magcapture™ exosome isolation kit(Wako).

FIG. 14f is a diagram showing a particle size distribution of exosomesisolated from the urine sample by Exo-Spin™ purification kit (CellGuidance System).

FIG. 15a is a scanning electron microscope (SEM) image of the exosomesisolated from the urine sample by the isolation chip of FIG. 1.

FIG. 15b is a transmission electron microscopy (TEM) image of theexosomes isolated from the urine sample by the isolation chip of FIG. 1.

FIG. 16 is a staining image showing protein contamination in theexosomes respectively isolated by the isolation chip (labeled asEI-Chip), the qEV™, the ExoQuick-TC™, the Magcapture™, and theExo-Spin™, after subjection to electrophoresis followed by silverstaining.

FIG. 17 is Western blot analysis of the exosomes isolated, by theisolation chip, from eleven urine samples of different cancer patients.

DETAILED DESCRIPTION

It will be appreciated that for simplicity and clarity of illustration,where appropriate, reference numerals have been repeated among thedifferent figures to indicate corresponding or analogous components. Inaddition, numerous specific details are set forth in order to provide athorough understanding of the embodiments described herein. However, itwill be understood by those of ordinary skill in the art that theembodiments described herein can be practiced without these specificdetails. In other instances, methods, procedures, and components havenot been described in detail so as not to obscure the related relevantfeature being described. Also, the description is not to be consideredas limiting the scope of the embodiments described herein. The drawingsare not necessarily to scale and the proportions of certain parts may beexaggerated to better illustrate details and features of the presentdisclosure.

The term “comprising,” when utilized, means “including, but notnecessarily limited to”; it specifically indicates open-ended inclusionor membership in the so-described combination, group, series, and thelike.

FIGS. 1 and 2 illustrate an embodiment of an isolation chip 10 adaptedfor isolation and purification of target particles from a liquid sample.The liquid sample can be a bioliquid such as plasma, serum, saliva,urine, and lavage. The target particles can be biological cells such ascirculating tumor cells (CTCs) or exosomes. The target particles canalso be other particles such as synthesized liposomes or nanospheres.

The isolation chip 10 includes a sample reservoir 13, a first chamber15, a second chamber 17, a first filtration membrane 14, and a secondfiltration membrane 16. The first chamber 15 and the second chamber 17are positioned at opposite sides of the sample reservoir 13. The firstchamber 15 is connected to the sample reservoir 13 by the firstfiltration membrane 14. The first chamber 15 includes a first outlet 152that connects the first chamber 15 to an ambient environment. The secondchamber 17 is connected to the sample reservoir 13 by the secondfiltration membrane 16. The second chamber 17 includes a second outlet172 that connects the second chamber 17 to the ambient environment.

In an embodiment, the sample reservoir 13 includes a reservoir substrate136, a first inner cover 132, and a second inner cover 134. Thereservoir substrate 136 is substantially U-shaped, and has a certainthickness. The first inner cover 132 and the second inner cover 134 arepositioned at opposite sides of the reservoir substrate 136. Thereservoir substrate 136, the first inner cover 132, and the second innercover 134 cooperatively define a receiving space (not labeled) toreceive the liquid sample. The first filtration membrane 14 is attachedto the first inner cover 132. The second filtration membrane 16 isattached to the second inner cover 134. Referring to FIG. 2, in anembodiment, each of the first inner cover 132 and the second inner cover134 define a through hole (not labeled). The first filtration membrane14 and the second filtration membrane 16 are fixedly received in thethrough holes of the first inner cover 132 and the second inner cover134, respectively. Furthermore, the sample reservoir 13 defines an inlet138 on the top. The liquid sample can be added to or removed from thesample reservoir 13 through the inlet 138.

In use, the liquid sample is added to the sample reservoir 13. Each ofthe first outlet 152 and the second outlet 172 is connected to a vacuumunit 30 (shown in FIG. 6). When the vacuum unit 30 generates a negativepressure in the first chamber 15 through the first outlet 152,compositions in the liquid sample that are smaller than the pores of thefirst filtration membrane 14 can enter the first chamber 15 through thefirst filtration membrane 14. When the vacuum unit 30 generates anegative pressure in the second chamber 17 through the second outlet172, compositions in the liquid sample that are smaller than the poresof the second filtration membrane 16 can enter the second chamber 17through the second filtration membrane 16. Since a negative pressure isalternately applied in the first chamber 15 and the second chamber 17,the compositions in the liquid sample can alternately flow through thefirst filtration membrane 14 and the second filtration membrane 16. Thisleaves the target particles that are larger than the pores of the firstfiltration membrane 14 and the second filtration membrane 16 in thesample reservoir 13. Furthermore, some of the target particles that areabsorbed on the first filtration membrane 14 and the second filtrationmembrane 16 can be flushed out under the negative pressure, therebyavoiding clogging of the first filtration membrane 14 and the secondfiltration membrane 16.

The first filtration membrane 14 and the second filtration membrane 16can be made of ceramic, plastic, or metal. In an embodiment, the firstfiltration membrane 14 and the second filtration membrane 16 can be madeof anodic aluminum oxide (AAO), polycarbonate, acetate fibers,polyethylene, polypropylene, polystyrene, and any combination thereof.The first filtration membrane 14 and the second filtration membrane 16can be made of a same material or different materials. Furthermore, thefirst filtration membrane 14 and the second filtration membrane 16 canhave a same average pore size or different pore sizes. In an embodiment,both the first filtration membrane 14 and the second filtration membrane16 are made of anodic aluminum oxide that have a high porosity and anaverage pore size.

The pore sizes of the first filtration membrane 14 and the secondfiltration membrane 16 can be varied according to the type of the liquidsample and the type of the target particles. In an embodiment, the poresizes of the first filtration membrane 14 and the second filtrationmembrane 16 are between 2 μm and 20 μm. Preferably, the pore sizes ofthe first filtration membrane 14 and the second filtration membrane 16are between 5 μm and 10 μm. More preferably, the pore sizes of the firstfiltration membrane 14 and the second filtration membrane 16 are 8 μm,thus the first filtration membrane 14 and the second filtration membrane16 can isolate CTCs from a plasma sample. In another embodiment, thepore sizes of the first filtration membrane 14 and the second filtrationmembrane 16 are between 5 μm and 200 μm. Preferably, the pore sizes ofthe first filtration membrane 14 and the second filtration membrane 16are between 10 μm and 100 μm. More preferably, the pore sizes of thefirst filtration membrane 14 and the second filtration membrane 16 are20 μm, thus the first filtration membrane 14 and the second filtrationmembrane 16 can isolate exosomes from a plasma sample.

When the surfaces of the first filtration membrane 14 and the secondfiltration membrane 16 are not modified, the isolation chip 10 may alsoisolate non-exosomal proteins that have similar sizes and densities asthose of the exosomes from the liquid sample. The non-exosomal proteinsinclude high density lipoproteins (HDLs), low density lipoproteins(LDLs), intermediate density lipoproteins (IDLs), very low densitylipoproteins (VLDL), and chylomicrons. In an embodiment, the surfaces ofthe first filtration membrane 14 and the second filtration membrane 16can be chemically modified by specific biological macromolecules, suchas antibodies, antigens, peptides, or chip base sequences, to allow theisolation chip 10 to isolate specified target particles.

In an embodiment, the first chamber 15 includes a first side cover 156facing away from the first inner cover 132. The first side cover 156 andthe first inner cover 132 cooperatively define the first chamber 15. Thefirst outlet 152 is defined in the first side cover 156. The secondchamber 17 includes a second side cover 176 facing away from the secondinner cover 134. The second side cover 176 and the second inner cover134 cooperatively define the second chamber 17. The second outlet 172 isdefined in the second side cover 176. Furthermore, each of the firstside cover 156 and the second side cover 176 can include an outletconnecting block 18. The outlet connecting block 18 defines a channel182 aligned with the first outlet 152 or the second outlet 172. Theisolation chip 10 can further include a chip base 19. The chip base 19closes ends of the first chamber 15 and the second chamber 17 oppositeto the inlet 138. The isolation chip 10 can have a symmetric or anasymmetric structure.

The reservoir substrate 136, the first inner cover 132, the second innercover 134, the first side cover 156, and the second side cover 176 canbe made of plastic, glass, metal, or composite materials. In anembodiment, the reservoir substrate 136, the first inner cover 132, thesecond inner cover 134, the first side cover 156, and the second sidecover 176 are made of polyethyleneimine (PEI) or poly(methylmethacrylate) (PMMA).

FIG. 3 illustrates an embodiment of a manufacturing method of theisolation chip 10. The method is provided by way of embodiments, asthere are a variety of ways to carry out the method. The example methodcan begin at block 31.

At block 31, the reservoir substrate 136, the first inner cover 132, thesecond inner cover 134, the first side cover 156, the second side cover176, the first filtration membrane 14, and the second filtrationmembrane 16 are provided.

At block 32, the first side cover 156 is connected to a side of thefirst inner cover 132 to form the first chamber 15.

At block 33, the first filtration membrane 14 is connected to the firstinner cover 132.

At block 34, the reservoir substrate 136 and the second inner cover 134are successively connected to a side of the first inner cover 132 facingaway from the first side cover 156, to form the sample reservoir 13.

At block 35, the second filtration membrane 16 is connected to thesecond inner cover 134.

At block 36, the second side cover 176 is connected to a side of thesecond inner cover 134 facing away from the reservoir substrate 136, toform the second chamber 17. The isolation chip 10 is obtained at a lowmanufacturing cost.

In an embodiment, the reservoir substrate 136, the first inner cover132, the second inner cover 134, the first side cover 156, the secondside cover 176, the first filtration membrane 14, and the secondfiltration membrane 16 are connected to each other by adhesive. Theadhesive can be ultraviolet-cured adhesive or silicone adhesive.

FIG. 4 illustrates another embodiment of an isolation chip 10′. In theisolation chip 10′, the reservoir substrate 136, the first inner cover132, and the second inner cover 134 are omitted. Thus, the manufacturingcost can further be decreased. In this embodiment, the first side cover156 includes a first protruding block 154. The first protruding block154 divides the first side cover 156 into a first cover portion 1561 anda second cover portion 1562 which are at opposite sides of the firstprotruding block 154. The second side cover 176 includes a secondprotruding block 174 facing the first protruding block 154. The secondprotruding block 174 divides the second side cover 176 into a thirdcover portion 1761 and a fourth cover portion 1762 which are positionedat opposite sides of the second protruding block 174. The first coverportion 1561, the third cover portion 1761, the first protruding block154, and the second protruding portion 174 cooperatively define thesample reservoir 13.

The isolation chip 10′ further differs from the isolation chip 10 inthat the isolation chip 10′ includes two chip bases, that is, a firstchip base and a second chip base (both labeled 19 in FIG. 3). The firstchip base 19 is connected to an end of the first side cover 156, andfaces the first protruding block 154. The first filtration membrane 14is connected between the first protruding block 154 and the first chipbase 19, and faces the second cover portion 1562. The second coverportion 1562, the first filtration membrane 14, and the first chip base19 cooperatively define the first chamber 15. The second chip base 19 isconnected to an end of the second side cover 176, and faces the secondprotruding block 174. The second filtration membrane 16 is connectedbetween the second protruding block 174 and the second chip base 19, andfaces the fourth cover portion 1762. The fourth cover portion 1762, thesecond filtration membrane 16, and the second chip base 19 cooperativelydefine the second chamber 17. In an embodiment, a gap 194 is definedbetween the first protruding block 154 and the second protruding block174. The liquid sample can flow out of the sample reservoir 13 throughthe gap 194, and further flow into the first chamber 15 or the secondchamber 17 through the first filtration membrane 14 or the secondfiltration membrane 16. In an embodiment, a surface of the firstprotruding block 154 facing the first chip base 19 defines a firstmounting groove 1540. A surface of the first chip base 19 facing thefirst protruding block 154 defines a second mounting groove (notlabeled). Opposite sides of the first filtration membrane 14 are fixedlyreceived in the first mounting groove 1540 and the second mountinggroove respectively. Similarly, a surface of the second protruding block174 facing the second chip base 19 defines a third mounting groove 1740.A surface of the second chip base 19 facing the second protruding block174 defines a fourth mounting groove (not labeled). Opposite sides ofthe second filtration membrane 16 are fixedly received in the thirdmounting groove 1740 and the fourth mounting groove respectively.

FIG. 5 illustrates another embodiment of a manufacturing method of theisolation chip 10′. The method is provided by way of embodiments, asthere are a variety of ways to carry out the method. The example methodcan begin at block 21.

At block 51, the first side cover 156, the second side cover 176, thefirst filtration membrane 14, and the second filtration membrane 16 areprovided.

At block 52, the first filtration membrane 14 is connected between thefirst protruding block 154 and the chip bases 19 of the first side cover156.

At block 53, the second filtration membrane 16 is connected between thesecond protruding block 174 and the chip bases 19 of the second sidecover 176.

At block 54, the first side cover 156 is connected to the second sidecover 176, the first protruding block 154 faces the second protrudingblock 174 and the two chip bases 19 face each other. Thus, the firstcover portion 1561, the third cover portion 1761, the first protrudingblock 154, and the second protruding portion 174 cooperatively definethe sample reservoir 13. The second cover portion 1562, the firstfiltration membrane 14, and the chip base 19 cooperatively define thefirst chamber 15. The fourth cover portion 1762, the second filtrationmembrane 16, and the chip base 19 cooperatively define the secondchamber 17. Thereby, the isolation chip 10′ is obtained.

FIG. 6 illustrates an embodiment of an isolation device 100 including amain device portion 101, an auxiliary device portion 102, and aninteraction device portion 103.

The main device portion 101 is configured to isolate and purify thetarget particles from the liquid sample. The main device portion 101includes the isolation chip 10 or 10′, a liquid provider 20, a vacuumunit 30, and a frequency converting unit 40.

The liquid provider 20 provides the liquid sample and a washing liquidinto the sample reservoir 13 of the isolation chip 10 or 10′. Referringto FIG. 7, the liquid provider 20 includes a liquid sample pool 210 forreceiving the liquid sample, a washing liquid pool 230 for receiving thewashing liquid, and a first valve 220. The first valve 220 isalternately switched to connect one of the liquid sample pool 210 andthe washing liquid pool 230. The first valve 220 can be anelectromagnetic valve or a rotary valve. When the first valve 220connects to the liquid sample pool 210, the liquid sample in the liquidsample pool 210 can be added to the sample reservoir 13. When the firstvalve 220 is switched to connect to the washing liquid pool 230, thewashing liquid in the washing liquid pool 230 can be added to the samplereservoir 13 to wash the isolation chip 10 or 10′. The washing liquidcan include a surfactant to wash away the proteins absorbed on surfacesof the isolation chip 10 or 10′. In another embodiment, the liquidprovider 20 can also be a pipette or a syringe. The liquid sample andthe washing liquid can thus be manually added to the sample reservoir13.

The vacuum unit 30 generates negative pressures in the first chamber 15and the second chamber 17 alternately. In an embodiment, the vacuum unit30 includes a first vacuum pump 310 and a second vacuum pump 320. Thefirst vacuum pump 310 is connected to the first outlet 152 of theisolation chip 10 or 10′. The second vacuum pump 320 is connected to thesecond outlet 172 of the isolation chip 10 or 10′.

The frequency converting unit 40 is electrically connected to the vacuumunit 30, and provides electric power to the vacuum unit 30. In anembodiment, the frequency converting unit 40 includes a frequencyconverter 410 and a second valve 420 connected to the frequencyconverter 410. The second valve 420 can be an electromagnetic valve or arotary valve. The second valve 420 is alternately switched to connectone of the first vacuum pump 310 and the second vacuum pump 320, tocause the vacuum unit 30 to alternately apply negative pressures in thefirst chamber 15 and the second chamber 17. That is, when the secondvalve 420 connects to the first vacuum pump 310, the frequency converter410 controls the first vacuum pump 310 to generate negative pressure inthe first chamber 15, corresponding to the left chamber in FIG. 9. Asshown by the arrows in FIG. 9, the compositions that are smaller thanthe pores of the first filtration membrane 14 can pass through the firstfiltration membrane 14 under the negative pressure. At the same time,the back flow of the liquid sample adjacent to the second filtrationmembrane 16 prevents any composition from accumulating in the pores ofthe second filtration membrane 16. Thus, clogging of the secondfiltration membrane 16 can be avoided. Then, the frequency converter 410controls the first vacuum pump 310 to stop operating, and the secondvalve 420 is switched to connect to the second vacuum pump 320. Thefrequency converter 410 controls the second vacuum pump 320 to applynegative pressures in the second chamber 17 corresponding to the rightchamber in FIG. 9. As shown by the arrows in FIG. 9, the compositionsthat are smaller than the pores of the second filtration membrane 16 canpass through the second filtration membrane 16 under the negativepressure. At the same time, back flow of the liquid sample adjacent tothe first filtration membrane 14 prevents any composition fromaccumulating in the pores of the first filtration membrane 14. Thus,clogging of the first filtration membrane 14 can be avoided. Then, thefrequency converter 410 controls the second vacuum pump 320 to stopoperating. The above procedures are repeated until complete isolation isachieved. Referring to FIG. 10a , in an embodiment, the negativepressures alternating between the first chamber 15 and the secondchamber 17 are caused by rectangular wave shaped pulse signals. Therectangular wave shaped pulse signals have an amplitude of −70 kpa and aperiod of 1 min. Since a sudden change of direction of the rectangularwave shaped pulse signals may cause damage to the first filtrationmembrane 14 and the second filtration membrane 16, FIGS. 10b and 10cdisclose another embodiment where shapes of the wave pulse signals canbe sine wave shaped or trapezoidal wave shaped. In other embodiments,since the plasma sample may have a large amount of proteins, the vacuumunit 30 can apply a positive pressure in one of the first or the secondchamber when applying a negative pressure in another one of the first orthe second chamber. By applying such positive pressure, the vacuum unit30 also improves the back flow at the first filtration membrane 14 orthe second filtration membrane 16, to further avoid clogging of thefirst and the second filtration membranes 14 and 16. In actual use, theamplitude, the period, and the total time durations of the negativepressures can be varied according to the type of the liquid sample.

Referring to FIGS. 11 and 12, in an embodiment, the main device portion101 can further include a mounting base 50 configured for receiving theisolation chip 10 or 10′. The mounting base 50 includes a bottom plate51, a mounting body 52, and two connecting plates 53 each mounted on thebottom plate 51. The mounting body 52 defines a receiving groove 520 forreceiving the isolation chip 10 or 10′. The mounting body 52 furtherincludes two sidewalls 521 opposite to each other. A top of each of thesidewalls 521 facing away from the bottom plate 51 defines a slot 522.When the isolation chip 10 or 10′ is received in the receiving groove520, each of the outlet connecting blocks 18 of the isolation chip 10 or10′ is received in one of the slot 522. Referring to FIG. 8b , each ofthe outlet connecting blocks 18 is thicker than each of the sidewalls521, so the outlet connecting block 18 protrudes from the slot 522 whenthe outlet connecting block 18 is received in the slot 522.

The connecting plates 53 are positioned at opposite ends of the mountingbody 52. Each of the first vacuum pump 310 and the second vacuum pump320 includes an air pipe 330 and a pipe connecting block 340 connectedto an end of the air pipe 330 facing away from the first vacuum pump 310or the second vacuum pump 320. The pipe connecting block 340 defines athird outlet 341 that is aligned with the air pipe 330. Each air pipe330 passes through one connecting plate 53, to position the pipeconnecting block 340 between the connecting plate 53 and the mountingbody 52. A spiral spring 331, positioned between the connecting plate 53and the pipe connecting block 340, surrounds each air pipe 330. When theisolation chip 10 or 10′ is received in the receiving groove 520, thefirst outlet 152 and the second outlet 172 are aligned with the thirdoutlet 341. Since each outlet connecting block 18 protrudes from theslot 522, the outlet connecting block 18 can push the pipe connectingblock 340 to move towards the connecting plate 53 to compress the spiralspring 331. The spiral spring 331 then rebounds to resist the isolationchip 10 or 10′ against the pipe connecting block 340. Air leakagebetween the isolation chip 10 or 10′ and the pipe connecting block 340is thus prevented. In an embodiment, the pipe connecting block 340includes an sealing ring 340 surrounding the third outlet 341. Thesealing ring 340 can further improve airtightness between the isolationchip 10 or 10′ and the pipe connecting block 340.

Referring to FIGS. 6 and 7, in an embodiment, the main device portion101 further includes a liquid collector 60. The liquid collector 60collects the target particles after isolation from the sample reservoir13. The liquid collector 60 can include a sampling needle that can beinserted into the sample reservoir 13 to collect the target particlesafter isolation.

Furthermore, referring to FIG. 7, the main device portion 101 furtherincludes a first liquid storage 350 and a second liquid storage 360. Thefirst liquid storage 350 is connected between the first vacuum pipe 310and the first outlet 152 of the isolation chip 10 or 10′, and isconnected to the first chamber 15 through the first outlet 152. Thesecond liquid storage 360 is connected between the second vacuum pipe320 and the second outlet 172 of the isolation chip 10 or 10′, and isconnected to the second chamber 17 through the second outlet 172. Thefirst liquid storage 350 and the second liquid storage 360 prevent theliquid sample from flowing into the first vacuum pump 310 and the secondvacuum pump 320.

The auxiliary device portion 102 is configured to ensure the isolationdevice 100 operates normally and efficiently. The auxiliary deviceportion 102 includes a detector 70 and a controller 80.

The detector 70 detects a liquid level of the liquid sample in thesample reservoir 13.

The controller 80 is electrically connected to the detector 70 and thefrequency converting unit 40. The controller 80 obtains the detectedliquid level, and determines whether isolation of the liquid sample isfinished according to the obtained liquid level and a first presetamount of the liquid sample. The obtained liquid level corresponds to aremaining amount of the liquid sample. The first preset amountcorresponds to an input amount of the liquid sample, which is usuallygreater than the remaining amount of the liquid sample. When isolationof the liquid sample is finished, the controller 80 controls thefrequency converting unit 40 to stop generating negative pressures inthe first chamber 15 and the second chamber 17. The controller 80 can bea central processing unit (CPU), a microprocessor, or other dataprocessor chip. In an embodiment, the controller 80 controls thefrequency converting unit 40 to generate negative pressures in the firstchamber 15 and the second chamber 17 according to preset pressure data.The controller 80 further controls a time interval for connecting thefirst valve 220 and the liquid sample pool 210 according to the firstpreset amount of the liquid sample, thereby allowing the liquid samplewith the first preset amount to be added to the sample reservoir 13. Thecontroller 80 further controls a time interval for connecting the firstvalve 220 and the washing liquid pool 230 according to a second presetamount of the washing liquid, thereby allowing the washing liquid withthe second preset amount to be added to the sample reservoir 13.

The interaction device portion 103 allows target particles isolationfrom the liquid sample to meet actual need. The interaction deviceportion 103 includes a user interface 90 for the user to input datarelated to the isolation processes through an input unit (for example, amouse or a keyboard) of the isolation device 100. That is, the user canpreset the data related to the isolation processes through the userinterface 90. In an embodiment, the data related to the isolationprocesses includes the first preset amount of the liquid sample, thesecond preset amount of the washing liquid, and the preset pressuredata. The preset pressure data includes the amplitude, the period, andthe total time durations of the negative pressures. The controller 80 isfurther electrically connected to the user interface 90. Thus, thecontroller 80 can obtain the input data from the user interface 90, andcontrol the frequency converting unit 40 or the liquid provider 20 tooperate accordingly.

In an embodiment, the interaction device portion 103 can further includea transmission interface 92 configured to connect the isolation device100 to a peripheral device (for example, a smart phone or a USB flashdisk). The isolation device 100 can transmit data related to theisolation processes to the peripheral device through the transmissioninterface 92. Thus, the user can review the data related to theisolation processes after sample isolation. The transmission interface92 can be a USB interface or a wireless interface.

The auxiliary device portion 102 further includes a memory 82. Thememory 82 stores an isolation control system 200. The isolation controlsystem 200 includes a number of modules, which are a collection ofsoftware instructions executable by the controller 80 to perform thefunction of the isolation control system 200. Referring to FIG. 8, theisolation control system 200 includes a second control module 202 and athird control module 203.

The second control module 202 controls the liquid provider 20 to providethe liquid sample and a washing liquid into the sample reservoir 13 ofthe isolation chip 10 or 10′. In an embodiment, the liquid provider 20includes a liquid sample pool 210 for receiving the liquid sample, awashing liquid pool 230 for receiving the washing liquid, and a firstvalve 220. The first valve 220 is alternately switched to connect one ofthe liquid sample pool 210 and the washing liquid pool 230. The secondcontrol module 202 controls the first valve 220 to connect to one of oneof the liquid sample pool 210 and the washing liquid pool 230, therebyallowing the liquid sample or the washing liquid to be added to thesample reservoir 13.

The third module 203 controls the vacuum unit 30 generates negativepressures in the first chamber 15 and the second chamber 17 alternatelythrough the frequency converting unit 40. In an embodiment, the vacuumunit 30 includes a first vacuum pump 310 and a second vacuum pump 320.The first vacuum pump 310 is connected to the first outlet 152 of theisolation chip 10 or 10′. The second vacuum pump 320 is connected to thesecond outlet 172 of the isolation chip 10 or 10′. The frequencyconverting unit 40 includes a frequency converter 410 and a second valve420 connected to the frequency converter 410. The third control module203 controls the second valve 420 to connect one of the first vacuumpump 310 and the second vacuum pump 320, to cause the vacuum unit 30 toalternately apply negative pressures in the first chamber 15 and thesecond chamber 17.

In an embodiment, the isolation device 100 further includes a liquidcollector 60. The second control module 202 further controls the liquidcollector 60 to collect the target particles after isolation from thesample reservoir 13.

In an embodiment, the isolation device 100 further includes a detector700. The detector 70 detects a liquid level of the liquid sample in thesample reservoir 13. The isolation control system 200 further includes afirst control module 201. The first control module 201 obtains thedetected liquid level from the detector 70, and determines whetherisolation of the liquid sample is finished according to the obtainedliquid level and a first preset amount of the liquid sample. Whenisolation of the liquid sample is finished, the first control module 201sends a stop command to the third control module 203. The third controlmodule 203 responds to the stop command, and control the frequencyconverting unit 40 to stop generating negative pressures in the firstchamber 15 and the second chamber 17.

In an embodiment, the first control module 201 further obtains thepreset pressure data, and sends a control command including the presetpressure data to the third control module 203. The third control module203 responds to the first control command, and controls the frequencyconverting unit 40 to generate negative pressures in the first chamber15 and the second chamber 17 according to preset pressure data. Thefirst control module 201 further obtains the first preset amount of theliquid sample, and sends a second control command including the firstpreset amount to the second control module 202. The second controlmodule 202 responds to the second control command, and controls a timeinterval for connecting the first valve 220 and the liquid sample pool210 according to the first preset amount, thereby allowing the liquidsample with the first preset amount to be added to the sample reservoir13. The first control module 201 further obtains the second presetamount of the washing liquid, and sends a third control commandincluding the second preset amount to the second control module 202. Thesecond control module 202 responds to the third control command, andcontrols a time interval for connecting the first valve 220 and thewashing liquid pool 230 according to the second preset amount, therebyallowing the washing liquid with the second preset amount to be added tothe sample reservoir 13

FIG. 13 illustrates an embodiment of an isolation method of targetparticles from liquid sample. The method is provided by way ofembodiment, as there are a variety of ways to carry out the method. Themethod can begin at block 131.

At block 131, the isolation chip 10 or 10′ is provided.

At block 132, the liquid sample is added to the sample reservoir 13 ofthe isolation chip 10 or 10′.

In an embodiment, the liquid sample is added to the sample reservoir 13by the liquid provider 20. The liquid sample can be added to the samplereservoir 13 through the inlet 138. To prevent the proteins fromabsorbing on the pores of the first filtration membrane 14 and thesecond filtration membrane 16, a surfactant and a PBS buffer are furtheradded to the sample reservoir 13. The surfactant can be Tween-20 orPluronic F68. The surfactant can have a weight percentage of about 5%.

At block 133, the first chamber 15 is evacuated through the first outlet152 to generate a negative pressure in the first chamber 15.

In an embodiment, before evacuating the first chamber 15, the firstoutlet 152 and the second outlet 172 are connected to the vacuum unit30. Then, the vacuum unit 30 evacuates the first chamber 15 through thefirst chamber 15, to cause the compositions having sizes which aresmaller than sizes of the pores of the first filtration membrane 14 toenter the first chamber 15 through the first filtration membrane 14.When the first chamber 15 has a small volume or when the negativepressure is switched too fast, the compositions can also enter the firstliquid storage 350 through the first outlet 152.

In one embodiment, before evacuating the first chamber 15, when theliquid sample is added to the sample reservoir 13 through the inlet 138,the inlet 138 can be closed. When the inlet 138 is closed, the back flowof the liquid sample adjacent to the second filtration membrane 16 canbe accelerated to avoid clogging of the second filtration membrane 16.

In other embodiments, since the plasma sample may have a large amount ofproteins, a positive pressure can be generated in the second chamber 17to further avoid clogging of the second filtration membrane 16.

At block 134, vacuuming of the first chamber 15 is stopped.

At block 135, the second chamber 17 is evacuated through the secondoutlet 172 to generate a negative pressure in the second chamber 17.

When the vacuum unit 30 evacuates the second chamber 17 through thesecond outlet 172, compositions which are absorbed on the firstfiltration membrane 14 may be separated from the first filtrationmembrane 14. Furthermore, the compositions having sizes which aresmaller than the sizes of the pores of the second filtration membrane 16can enter the second chamber 17 through the second filtration membrane16. When the second chamber 17 has a small volume or when the negativepressure is switched to fast, the compositions can also enter the secondliquid storage 360 through the second outlet 172. In other embodiments,the blocks 134 and 135 can also be performed simultaneously.

In other embodiments, since the plasma sample may have a large amount ofproteins, a positive pressure can be generated in the first chamber 15to further avoid blocking and clogging of the first filtration membrane14.

At block 136, vacuuming of the second chamber 17 is stopped.

Then, the blocks 133 to 136 can be repeated for a number of times tofurther remove the compositions having sizes which are smaller than thesizes of the pores of the first filtration membrane 14 and the secondfiltration membrane 16, and causing the target particles to remain inthe sample reservoir 13.

At block 137, the washing liquid is added to the sample reservoir 13.Then, the blocks 133 to 136 can be repeated for a number of times towash the isolation chip 10 or 10′.

Using the above method to isolate and purify exosomes from a urinesample of 10 mL, a high yield of exosomes was obtained within 30 min.Furthermore, exosomes were also isolated and purified, respectively byqEV™, ExoQuick-TC™, MagCapture™, and Exo-Spin™, from the same urinesample. Then, the exosomes isolated by different approaches were testedby a particle size analyzer (Malvern). Referring to FIGS. 14a to 14f ,the exosomes isolated by different approaches showed similar particlesize distributions in the range of 30-150 nm, which matched the actualparticle size distribution of the exosomes.

The exosomes isolated by the isolation chip 10 were observed with SEMand TEM. Referring to FIGS. 15a and 15b , the exosomes after isolationhad good integrity and high purity.

Since HDLs, LDLs, IDLs, VLDL, and chylomicrons, which have similar sizeand density with respect to the exosomes, are difficult to be removed,to study the purity of the exosomes, the original urine sample and theexosomes obtained by different approaches were subjected toelectrophoresis followed by silver staining, to identify the proteinstherein. Referring to FIG. 16, the original urine sample showed strongsignals, which indicated a large amount of proteins contained in theoriginal urine sample. After isolation and purification by qEV™ andExo-Spin™, the exosomes still showed strong signals, indicating that theexosomes still contain a large amount of proteins. After isolation andpurification by MagCapture™ and ExoQuick-TC™, the majority of proteinswere removed and therefore the exosomes showed much weaker signals.After isolation and purification by the isolation chip 10 (labeled asEI-Chip in the figure), the exosomes showed signals much weaker thanthose isolated by qEV™ and Exo-Spin™, and slightly higher than thoseisolated by MagCapture™ and ExoQuick-TC™. That is, the exosomes isolatedby purified by the isolation chip 10 have high purity. The isolationchip 10 has competitive performance in isolating exosomes from the urinesample compared to the commercial products.

The fluid sample of a cancer patient may be different from a healthyfluid sample, and the fluid samples of different cancer patients alsohave different properties. To make sure that the isolation chip 10 canalso be use to successfully isolate the exosomes from the fluid samplesof different cancer patients, urine samples from 11 prostate cancerpatients, each with 10 mL, were collected. The exosomes wererespectively isolated from the urine samples by the isolation chip 10.The amount of proteins in the exosomes from different urine samples wasmeasured with Nanodrop, and the results were shown in Table 1.

TABLE 1 Urine samples Amount of proteins (mg/mL) 1 0.275 2 0.731 3 3.0994 0.826 5 0.321 6 0.165 7 0.998 8 1.112 9 2.21 10 0.624 11 0.944

As shown in Table 1, the exosomes from 8 out of 11 urine samples have aprotein concentration lower than 1 mg/mL after isolation andpurification. Thus, the exosomes from most of the urine samples have asmall amount of proteins, and thus have high purity.

Furthermore, as shown in FIG. 17, Western blot analysis of the exosomesfrom 7 out of 11 urine samples revealed a presence of CD81 and CD9(exosome marker), and 2 of 11 urine samples revealed a presence of oneof CD81 and CD9. Thus, the isolation chip 10 can be use to successfullyisolate the exosomes from the fluid samples of different cancerpatients.

Moreover, to test reproducibility and robustness of the isolation chip10, the exosomes were repeatedly isolated from the same fluid samplesfor 5 times by the same isolation chip 10. The amount of proteins in theexosomes from different urine samples was measured with Nanodrop. Theresults showed that the amount of proteins in the exosomes has acoefficient of variation smaller than 5%, compared to the data ofTable 1. Thus, the isolation chip 10 has a good reproducibility forexosome isolation and purification. Furthermore, the exosomes wererespectively isolated from the same fluid samples by 50 isolation chip10, and the failure rate is smaller than 5%, which also indicates thatthe isolation chip 10 has a good reproducibility for exosome isolationand purification.

The embodiments shown and described above are only examples. Therefore,many commonly-known features and details are neither shown nordescribed. Even though numerous characteristics and advantages of thepresent technology have been set forth in the foregoing description,together with details of the structure and function of the presentdisclosure, the disclosure is illustrative only, and changes may be madein the detail, including in matters of shape, size, and arrangement ofthe parts within the principles of the present disclosure, up to andincluding the full extent established by the broad general meaning ofthe terms used in the claims. It will, therefore, be appreciated thatthe embodiments described above may be modified within the scope of theclaims.

What is claimed is:
 1. An isolation chip for isolation and purificationof target particles from a liquid sample, comprising: a sample reservoirconfigured for receiving the liquid sample; a first filtration membranecomprising pores of sizes smaller than sizes of the target particles; asecond filtration membrane comprising pores of sizes smaller than thesizes of the target particles, wherein the sample reservoir is disposedbetween the first filtration membrane and the second filtrationmembrane; a first chamber connected to the sample reservoir through thefirst filtration membrane, the first chamber defining a first outletconnecting the first chamber to an ambient environment; and a secondchamber connected to the sample reservoir through the second filtrationmembrane, the second chamber defining a second outlet connecting thesecond chamber to the ambient environment; wherein the sample reservoircomprises a reservoir substrate, a first inner cover, and a second innercover, each of the first inner cover and the second inner cover is at aside of the reservoir substrate and opposite to each other, each of thefirst inner cover and the second inner cover define a through hole, eachof the first filtration membrane and the second filtration membrane isfixedly received in each of the through holes, the first chambercomprises a first side cover facing away from the first inner cover, thefirst side cover and the first inner cover cooperatively define thefirst chamber, the first outlet is defined in the first side cover, thesecond chamber comprises a second side cover facing away from the secondinner cover, the second side cover and the second inner covercooperatively define the second chamber, and the second outlet isdefined in the second side cover.
 2. The isolation chip of claim 1,wherein each of the first side cover and the second side cover comprisesan outlet connecting block, each of the outlet connecting blocks definesa channel aligned with each of the first outlet and the second outlet.3. The isolation chip of claim 1, wherein the sample reservoir definesan inlet on top.
 4. The isolation chip of claim 3, further comprising achip base to close ends of the first chamber and the second chamberopposite to the inlet.
 5. The isolation chip of claim 1, wherein thereservoir substrate, the first inner cover, the second inner cover, thefirst side cover, the second side cover are made of poly ethyleneimineor poly(methyl methacrylate).
 6. The isolation chip of claim 1, whereinthe first side cover comprises a first protruding block, the firstprotruding block divides the first side cover into a first cover portionand a second cover portion which are at two opposite sides of the firstprotruding block, the second side cover comprises a second protrudingblock facing the first protruding block, the second protruding blockdivides the second side cover into a third cover portion and a fourthcover portion which are at opposite sides of the second protrudingblock, the first cover portion, the third cover portion, the firstprotruding block, and the second protruding block cooperatively definethe sample reservoir, a first chip base is connected to an end of thefirst side cover and faces the first protruding block, the firstfiltration membrane is connected between the first protruding block andthe first chip base, and faces the second cover portion, the secondcover portion, the first filtration membrane, and the chip basecooperatively define the first chamber, a second chip base is connectedto an end of the second side cover and faces the second protrudingblock, the second filtration membrane is connected between the secondprotruding block and the second chip base and faces the fourth coverportion, the fourth cover portion, the second filtration membrane, andthe second chip base cooperatively define the second chamber.
 7. Theisolation chip of claim 6, wherein a gap is defined between the firstprotruding block and the second protruding block, a flow path of theliquid sample is defined from the sample reservoir to each of the firstand the second chambers through the gap.
 8. The isolation chip of claim6, wherein a surface of the first protruding block facing the first chipbase defines a first mounting groove, a surface of the first chip basefacing the first protruding block defines a second mounting groove, twoopposite sides of the first filtration membrane are fixedly received inthe first mounting groove and the second mounting groove respectively, asurface of the second protruding block facing the second chip basedefines a third mounting groove, a surface of the second chip basefacing the second protruding block defines a fourth mounting groove, andtwo opposite sides of the second filtration membrane are fixedlyreceived in the third mounting groove and the fourth mounting grooverespectively.
 9. The isolation chip of claim 1, wherein each of thefirst filtration membrane and the second filtration membrane is made ofanodic aluminum oxide.
 10. A manufacturing method of an isolation chipfor isolation and purification of target particles from a liquid sample,the manufacturing method comprising: providing a reservoir substrate, afirst inner cover, a second inner cover, a first side cover, a secondside cover, a first filtration membrane, and a second filtrationmembrane, the first side cover defining a first outlet, the second sidecover defining a second outlet, the first filtration membrane and thesecond filtration membrane comprising pores of sizes smaller than sizesof the target particles; forming a first chamber by connecting the firstside cover to a side of the first inner cover, the first outletconnecting the first chamber to an ambient environment; connecting thefirst filtration membrane to the first inner cover; forming a samplereservoir by successively connecting the reservoir substrate and thesecond inner cover to a side of the first inner cover facing away fromthe first side cover, thereby forming a flow path from the first chamberbeing to the sample reservoir through the first filtration membrane;connecting the second filtration membrane to the second inner cover,wherein the sample reservoir is disposed between the first filtrationmembrane and the second filtration membrane; and forming a secondchamber by connecting the second side cover to a side of the secondinner cover facing away from the reservoir substrate, thereby forming aflow path from the second chamber to the sample reservoir through thesecond filtration membrane, and the second outlet connecting the secondchamber to the ambient environment.
 11. The manufacturing method ofclaim 10, wherein the reservoir substrate, the first inner cover, thesecond inner cover, the first side cover, the second side cover, thefirst filtration membrane, and the second filtration membrane areconnected to each other by adhesive.
 12. The manufacturing method ofclaim 11, wherein the adhesive is ultraviolet-cured adhesive or siliconeadhesive.
 13. A manufacturing method of an isolation chip for isolationand purification of target particles from a liquid sample, themanufacturing method comprising: providing a first side cover comprisinga first protruding block and a first chip base, and a second side covercomprising a second protruding block and a second chip base, wherein thefirst protruding block divides the first side cover into a first coverportion and a second cover portion which are at two opposite sides ofthe first protruding block, the second protruding block divides thesecond side cover into a third cover portion and a fourth cover portionwhich are at opposite sides of the second protruding block, the firstchip base is connected to an end of the first side cover and faces thefirst protruding block, the second chip base is connected to an end ofthe second side cover and faces the second protruding block, the firstside cover defining a first outlet, the second side cover defining asecond outlet; providing a first filtration membrane and a secondfiltration membrane, each of the first filtration membrane and thesecond filtration membrane having pores of sizes smaller than sizes ofthe target particles; connecting the first filtration membrane betweenthe first protruding block and the first chip base; connecting thesecond filtration membrane between the second protruding block and thesecond chip base; forming a sample reservoir defined between the firstcover portion, the third cover portion, the first protruding block, andthe second protruding block; forming a first chamber defined between thesecond cover portion, the first filtration membrane, and the first chipbase; and forming a second chamber defined between the fourth coverportion, the second filtration membrane, and the second chip base;wherein the first chamber is connected to the sample reservoir throughthe first filtration membrane, and the second chamber being connected tothe sample reservoir through the second filtration membrane.